PROTEIN FUSIONS FOR THE TRANSLOCATION OF APO-PROTEIN INTO THE PERIPLASMIC SPACE

Abstract: A method of protein synthesis which comprises providing a genetic unit comprising a nucleotide sequence coding for a pre-form apo-protein. The pre-form apo-protein is synthesized in a cytoplasmic region of a cell and translocated to the periplasmic region of the cell for conversion to a corresponding holo-protein.

Description: PROTEIN FUSIONS FOR THE TRANSLOCATION OF APO-PROTEINE INTO THE PERIPLASMIC SPACE The present invention is concerned with protein synthesis and in particular with the production of recombinant fusions between cytochrome b, and foreign proteins which can be translocated out of the cell into the penplasmic space of, for example, Eschenchia coll. The mechanism by w hich a CN toplasmically-synthesised (recombinant) protein is translocated into the penplasmic space of Eschenchia co is known to occur by the workings of the signal hypothesis (see review by Stardler. J .A. and Silhavy, T.J.. 1990, Methods in Enzymology, 167- 187) Main such proteins are initially synthesised as precursor forms carrying extensions at their ammo termini known as signal sequences (peptides). The signal sequence has the information required tor selective translocation of the passenger part of the protein molecule across the cvtoplasmic membranes of the bacteria. The signal is cleaved off soon after the penplasmically-deposited protein has gained its native biological fold and function. Using this approach we have developed a bacterial system which allows production of chimenc or fusion forms ot coloured proteins. These proteins are secreted into the peπplasm where they are afforded greater protection against potential degradation. According to a first aspect of the present invention there is provided a method of protein synthesis which comprises provision ot a genetic unit comprising a nucleotide sequence coding for a pre-form ot an apo-protein. synthesizing said pre-form apo-protein in a cytoplasmic region of a cell, and translocating said synthesised pre-form apo-protein to the penplasmic region of said cell, so as to permit constitution in said periplasmic region of a signal-processed apo-protein which can then be converted to the corresponding holo-protein. According to a second aspect ot the present invention, there is provided a genetic precursor unit w hich comprises a nucleotide sequence coding for a pre-form apo- protein. said nucleotide sequence being such that said pre-form apo-protein is translocatable from a cvtoplasmic region ot a cell to the penplasmic region ot said cell, so as to permit constitution in said penplasmic region ot a processed apo-protein and conversion into a corresponding holo-prote The genetic unit is preferably suitable for expression in the cvtoplasmic region of a bacterial host cell, such as an E. coli host cell. The preferred E. coli strain is TB- 1. [F ara Δ (\ac-proAB) rps φ 80d lacZ Mlδ hsdRH (rk+ m ~k)] or N4830-1 [f-suo hisXlv-galK- (λchlD-pg])(\Eam N+ CI857 H I )] . A preferred apo-protein comprises a cvtoplasmic cytochrome, which typically comprises the soluble core domain of cytochrome b^ of liver endoplasmic reticulum. Cytochrome b of the endoplasmic reticulum of mammalian liver is a well characterised, small haemoprotein of 16.7 kD which plays a central role in a variety of electron transfer reactions related to fatty acid desaturation. redox cycling of oestrogen and reduction of cytochrome P-450 reductase. The hepatic cytochrome b ^ , is composed of two domains, the above-described soluble, enzymatically active, haem-containing globular core (b 3 ) of about 12 kD and a smaller carboxy terminal tail anchored in the microsomal membrane. Within the tail portion, a stretch of 23 hydrophobic amino acid residues, also definable as an "insertion" sequence, autonomously and post-translationally integrates cytochrome b s into the lipid bilayer of the endoplasmic reticulum such that the active core domain is laterally disposed from the reticulum facing the cytoplasm. It is preferred that the genetic unit further comprises a nucleotide sequence which codes for an amino-terminal signal peptide (the pre region of pre-form apo-protein) which is recognised by the cell to direct the pre-form apo-protein to the cvtoplasmic membrane and thence subsequently translocated into the periplasm of the cell. The signal peptide typically comprises E.coli alkaline phosphatase. which is advantageously in linkage with cytochrome b s (apo-protein). The method therefore preferably further comprises the step of directing the apo-prote to the cvtoplasmic membrane, under the direction of the signal peptide, so as to facilitate the above-described translocation of the pre-form apo- protein from the cytoplasm to the periplasmic space of the bacterium. In this preferred embodiment of the present invention where the genetic unit further comprises a nucleotide sequence which codes for an amino-terminal signal peptide. the invention can be used to investigate signal peptidase which in vivo proteolytically cleaves N-terminal signal peptides. Many proteins which are synthesised in the cytosol have to be targeted to various extracellular and subcellular locations This involves translocation across one or more lipid bilayers impermeable to large hydrophihc molecules A feature of most translocated proteins is that t ev are svnthesised as precursor forms carrying N-terminal signal sequences, which as discussed above are proteolytically cleaved by signal peptidase, either during or immediately after transport ot the passenger protein across the biological membrane. Signal peptidase is highly specific as it cleaves only preprotein precursors. Although lacking sequence homologies. signal sequences are functionally highly conserved. Despite significant variations in their lengths, all signals have three definable regions, namely an N-terminal region carrying one or tw o basic residues, a middle core encompassing hydrophobic residues and a terminal region specifying the cleavage site The conventional assay tor signal peptidase has until now relied on the use of natural precursor proteins which are synthesised in a cell-free translation system in a highly radioactive form. The extent of processing is monitored by incubating the radio-labelled substrate with either membranes or signal peptidase and monitoring the processed bands by autoradiography following gel electrophoresis The assay is not only time-consuming and expensive but suffers from the use ot minuscule quantities (tmol) of substrates in relation to enzyme quantities, with inevitable contamination by many cellular components derived from the use of in vuro synthesis systems We have now overcome the above problems, and there is further provided by the present invention an assay system tor signal peptidase, which assay system composes: (a) a genetic precursor unit hich comprises a nucleotide sequence coding for an apo- protein and an amino-terminal signal peptide. the nucleotide sequence being such that the apo-protein is translocatable from a cytoplasmic region ot a cell to the penplasmic region of the cell, so as to permit constitution in said penplasmic region ot a processed apo-protein: and (b) signal peptidase. a source thereof or a precursor therefor, wherein the signal peptidase can effect proteolytic cleavage of the ammo terminal signal peptide from the apo-protein. The genetic unit ot the assay system is substantially as hereinbefore descnbed and is advantageously suitable tor expression in a bacterial medium such as E.coli and the assay system preferably further comprises a host medium such as E.coli substantially as hereinbefore described. Preferably the assa_\ system further comprises means for monitoring the proteolytic cleavage of the ammo terminal signal peptide from the apo-protein. The monitoring means typically comprises means tor separating the cleaved apo-protein from the pre-form apo-protein, and means for spectrophotometπcally monitoring the cleaved apo- protein following supplementation thereof with exogenous haem. Typically the pre-form apo- protein is phase-separated from the cleaved apo-protein. the pre-form only being soluble as an aggregate in the presence ot a detergent, the cleaved apo-protein being water soluble. The provision ot such an assay system is advantageous in investigating the mechanistic and structural properties ot signal peptidase A particularly useful application of an assay system according to the present invention is in the identification of inhibitors of signal peptidase which can subsequently be used as probes in investigating the reaction mechanism of signal peptidase. There is further provided by the present invention a method of identifying inhibitors to signal peptidase. which method comprises providing an assay system substantially as described above, introducing a test material into cells of a host medium used in the assay system, and monitoring the effect ot said test material on a selected activity of the signal peptidase. Advantageously, the identification method involves monitoring the cleavage of the amino-terminal signal peptide from the apo-protein by spectrophotometnc means substantially as hereinbefore described. Preferably the test material comprises any of the following: a consensus signal peptide (which typically comprises a 19 residue peptide). one or more transition metal ions, and carboxy-modi tying reagents Signal peptidase is a known crucial rate limiting step in the pathway of protein secretion and can offer a locus for control or regulation of an essential biofunction, namely protein export. Thus the identification of a signal peptidase inhibitor can provide a tool to understand the reaction mechanism of signal peptidase. and also, more importantly, a means to regulate growth ot microorganisms and protein secretory disorders in higher organisms. The invention allow s the observ ation of the effects ot competitive inhibition by selected inhibitors in order to identity the precise region of signal sequence that is essential for recognition/cleavage by signal peptidase. There is further prouded by the present invention a method of recognising an active binding site of signal peptidase. w hich method comprises contacting the signal peptidase with at least on photoacm atable form of a radio-labelled (typically radioiodinated) inhibitor ot the peptidase, and identify ing the binding location of the inhibitor to the peptidase. There is further provided by the present invention, a test matenal identified as an inhibitor to signal peptidase. tor use in modulating in vivo protein secretion. The test material may advantageously comprise a synthetic inhibitor of signal peptidase. and has beneficial applications as a biocidal agent, regulating secretory disorders in mammalian cells, control ot plant growth and the like. The genetic unit typically comprises a plasmid having a vector as shown in any of Figures 1 to 3, or a DNA sequence as shown in any of Figures 4 to 6 (SEQ ID NOS 1 to 3. respectively). The most significant features of the plasmid sequences are as follows: a) a native pho promoter for transcnptional control. The pho promoter is available coded tor by the plasmids pSEC-cyt w hich is inducible by growth of the bacteria in a phosphate-limited medium. b) a signal sequence coding tor alkaline phosphatase as hereinbefore described: and c) a soluble cytochrome b, core gene sequence. There is further provided by the present invention a genetic unit having a vector substantially as illustrated in any of Figures 1 to 3. or a DNA sequence as illustrated in any of Figures 4 to 6 (SEQ ID NOS 1 to 3 respectively), which is capable of generating translocated forms non-fusion (Figure 1 or 4) or fusion (chimeπc) forms of coloured cytochrome b, (Figures 2.5 and 3.6) The foreign protein can be tagged to the cytochrome b s protein at its carboxy (C) (Figure 2.5 ) or ammo (N) terminus (Figure 3,6). The pho promoter is typically capable ot minimising the lethality of the expressed chimera to the host by maintaining the expression of chimera under the tight transcnptional control of the native pho promoter, in plasmids pSEC-cyt/C and pSEC-cyt/N. inducible only by growth of the bacteria in a phosphate limited medium. In pSEC-cyt/C fusions are introduced bv linkage ot a foreign DNA sequence at the carboxy terminus of cytochrome b. gene through the unique Be II sue In pSEC-c /N fusions are introduced immediately at the N-terminus ot tochrome b, but after the signal sequence, through the unique Bam l and/or Pstl sites The method ot expression of the pre-form cytochrome b, or its chimeπc derivative typically involves initiation ot transcription, and hence protein synthesis, by control ot the incubation conditions ot the host medium Typically the initiation stage comprises cultivation of the bacterial host a phosphate-limited medium (0. 1 mM phosphate), and spectrophotometnc or visual monitoring (transformation of the colour of the bacterial cells from a grey to pink colour) ot the synthesis cytochrome b, (processed) or its chimera (processed) in E.coli harbouring pAA-cyt or pSEC-cyt. respectively, under the transcnptional control ot the pho promoter It is further preferred that the method involves translocation of a prosthetic group from the cvtoplasmic region ot the cell to the penplasmic region, for combination with the signal-processed apo-torm of the chimeπc protein in the penplasmic region to form the holo-protein as hereinbefore described. It is preferred that the apo-protein is first translocated to the penplasmic region, followed by subsequent translocation of the prosthetic group, such that the apo-protein can act as a sink for the latter. In the case where the apo- protein comprises the cytochrome b- soluble core, the prosthetic material compnses the hae (protoporphyπn IX) prosthetic group. The penplasmic accrual of the apo cytochrome b 5 core is thought to trigger the subsequent translocation of the haem-prosthetic group. It is envisaged that a prosthetic material may be present in the penplasmic region ot the cell, although this does not appear to be the case with cytochrome b 5 due to the observed build up of penplasmic apo-cytochrome b, and ensuing time lag preceding the appearance of penplasmic holo-cytochrome b-. Production of the exported peπplasmically-localised haemoprotein can typically exceed 6 mg/litre of culture after five hours induction. Mutated forms of the cytochrome b s have been developed which can enhance exportation ot the mature forms by more than tenfold The approach may be useful tor the production and secretion of coloured forms and fusions in eukarvotic cells. The method according to the inv ention typically further comprises monitoring of protein synthesis by observing the v isual transformation of the bacterial cells from a grey translucent to a bright pink colour This colour change is due to the expression of the cytochrome b s gene. The monitoring may be carried out by spectrophotometnc means where an accurate analysis of the extent of protein synthesis is required, so allowing the protein synthesis to be terminated when sufficient protein has been synthesised. The above-described method allows a prokaryotic signal sequence to be appended with cytochrome b,. such that this natural cytoplasmic protein of eukaryotic origin is transformed into a secretory form in EΛ OII Furthermore, the expression of the eukaryotic gene encoding the pre-form apo-protein or its chimera, synthesised in the cytoplasm, is such that the post-translocationally processed component in the peπplasm gives rise to the holo- protein or its fusion counterpart. According to the present invention there is further provided a proteinaceous material comprising a pre-form apo-prote w hich is synthesised in the cytoplasmic region of a cell, the pre-torm apo-protein being translocatable to the penplasmic region of said cell, so as to allow constitution in said penplasmic region of a correspondingly processed apo- protein, the binding of a prosthetic group (typically the haem group) to the apo-protein forms the holo-protein. There is further provided by the present invention an apo-protein substantially as hereinbefore described, in combination w ith a signal peptide for selective translocation (export) or secretion of said apo-protein from the cytoplasmic to the penplasmic region of a cell, so as to allow constitution in the penplasmic region of a corresponding holo-protein from said apo-protein. According to yet a further aspect of the present invention there is provided a holo-protein constituted in the penplasmic region of a cell from an apo-protein, or by a method, substantially as hereinbefore described There is further provided a holo-protein derived from a genetic precursor unit, substantially as illustrated with reference to any of Figures 4 to 6 The aDove-descπbed apo- and holo-proteins typically respectively comprise the apo- and holo- forms of cytochrome b. or its respective N- or C-terminally fused chimeπc forms. The fusions can be generated at either the ammo or carboxy terminus of the secreted recombinant protein There is further provided by the present invention an E.coli host medium containing a genetic precursor unit substantially as hereinbefore described. There is further provided a kit for use in protein synthesis, which kit comprises: a) a bacterial host medium substantially as hereinbefore described; and b) genetic material comprising a nucleotide sequence coding for a pre-form apo-protein, such that said pre-form apo-protein coded for by said nucleotide sequence is translocatable from the cytoplasmic to the periplasmic region of a cell of the host medium, so as to allow constitution in the periplasmic region of a holo-protein from the apo-protein. The kit typically comprises a bacterial host medium, such as E.coli, together with genetic material comprising precursor constituent genes suitable to be included in a genetic unit substantially as hereinbefore described. An example of a typical synthesis of a fusion protein according to the present invention will now be described. A gene coding for a foreign protein or peptide was fused in a reading frame from either (i) the carboxy-terminus or (ii) the N-terminus of the soluble core of cytochrome b The foreign gene was ligated between either (i) the unique Bcli restriction site and the downstream polylinker site or (ii) the unique Psi\ and Ba Hl site located between the signal sequence and cytochrome b s . E.coli TB- 1 (Lac ) cells were then transformed with the ligation mixture. Transformants were plated out on Luria broth agar ( 1.5 % (w.v)) plates containing ampicillin (75μg/ml), isopropyl- -D-galactopyranoside (IPTG) (100μM),5-bromo- 4-chloro-3-indolyl- 3-D-galactoside (X-gal) (20μg/ml). Transformant colonies bearing potential inserts were white in colour whereas non-recombinants were blue in colour. If required, the recombinant plasmids could be further verified by restriction mapping. For induction, a starter bacterial culture cultivated to saturation in LB/ampicillin was applied at a 2 % (v/v) inoculum into MOPS medium. The cultures were grown with shaking at 100 revs/min at 35°C for 5 to lOh. The following table indicates the composition of the MOPS medium. All solutions were sterilised by autoclaving or filter sterilisation. The final concentration of phosphate in the MOPS medium was about O. l mM. Component % (v/v) in MOPS Water 74.5 M 20 O* 0.2 P a 0.01 S a 0. 1 VFCAA J 2 20% glucose' 1 0.05 % thiamine Bl ' 0.2 25mg/ml ampicillin 1 0.3 M, 4.2 % (w/v) MOPS buffer. 0.4 % (w/v) Tric e. 1 .46 % NaCl, 0.8% KOH, 0.2555 % NH C1 O. 5.36% MgC , 0. 16% HC1. 0. 1 % FeCl,. 0.00368% CaCl,.2H,O. 0.00128 % H,BO„ 0.0008 % MnCU.4H,O, 0.00036% CoCΪ 2 .H,O, 0.00008 % CuC1..2HX>. 0.0068 % ZnCK. 0.012 1 % Na,MoO 4 .2H 2 O. P, 1.0M KH 3 PO 4 S. 0.276M K,SO 4 VFCAA. 7.5 % vitamin-free Casamino acids ', autoclaved prior to use ', filter sterilised prior to use Positive expression of the fusion protein was indicated by pink colour of the bacterial cells obtained by centrifugation at 5.000 x g for 5 min. The protein was extracted from the bacteria by the following "osmotic shock" method: (i) The cells (derived from a 30 ml culture) were gently suspended in 1 ml of 20% sucrose. 0.3 M Tris-HCl (pH 8), I mM Na ? EDTA )(STE) and incubated at room temperature (22°C) for 10 min. (ii) The cells were then harvested as pellet by centrifugation at 5.000 x g for 6 min and thoroughly resuspended in residual STE volume. (iii) The plasmolysed cells were osmotically shocked by rapidly suspending in ice-cold 0.5mM MgC and left on ice for 10 min. Centrifugation at 10.000 x g for 10 min yielded supernatant comprising the periplasmic fraction. Pink coloration of the supernatant implied that fusion protein had been efficiently extracted from the periplasmic space. The quantity ot the recombinant protein was monitored by subjecting a fraction of the extract to a spectral scan from 350 to 50ϋnm. The oxidised cytochrome was identifiable by the rise in absorbance at 413nm. Often a significant pool of the apo-protein may be present. This may be converted to holo cytochrome by addition of haem to the supernatant to a final concentration ranging from 5 to 20μM: an equivalent amount of haem was added to the blank cuvette to counteract the absorbance rise by excess haem in the sample. The amount of protein may be calculated from the mM extinction coefficient of the oxidised cytochrome b, of 1 15 and 413nm. The present invention ill now be further illustrated, by way of example only, by reference to the accompanying Figures 1 to 6. wherein: Figures 1 .2 and 3 illustrate expression vectors pAA-cyt, pSEC-cyt/C and PSEC-cyt/N of a genetic unit according to the present invention: Figures 4-6 illustrate the genetic sequences of the vectors respectively illustrated in Figures 1 -3 (SEQ ID NOS 1 to 3. respectively); and Figures 7-9 schematically illustrate modifications of plasmids according to the present invention, which involve cloning, expression and isolation of the chimeric proteinaceous material. Figure 1 illustrates plasmid PAA-cyt constructed for the periplasmic production of non-fusion, secreted holo-cytochrome b,. Figure 2 illustrates the plasmid PSEC-cyt/C constructed for linkage at the 3' terminus (C-terminus) of the soluble cytochrome b s core gene with a foreign gene sequence, according to the present invention. Figure 3 illustrates the plasmid PSEC-cyt/N for linkage at 5' terminus, between the C-terminus of alkaline phosphatase signal and the N-terminus of cytochrome b 5 of the cytochrome b=, core gene, according to the present invention. Referring now to Figures 1 to 3. the essential features of the illustrated vector are as follows: Pho, Pho promoter activated in bacteria grown on phosphate limited medium; S/D, Shine Dalgarno sequence for translation initiation of alkaline phosphatase signal: SS, alkaline phosphatase signal sequence gene encoding 21 residue signal including the first arginine residue of mature alkaline phosphatase: C Y. soluble core of cytochrome b : T. tail of the native cytochrome b .: Lac, Lactose operon promoter: and PLS. polylinker sites: EcoRI, ( Hind 111). Sad. Kpnl. S al, BamHl, Xbal. Sail, Hindi. BspMl. Psrl. Spin. Hi dUl. (In the polylinker sites. EcoRI and Hindlll can be in the reverse order to that shown. ) SS-cyr/C, codes for the complete precursor of the cytochrome b 5 including the tail portion of the haemoprotein. Referring now to Figure 7. the figure illustrates the scheme of appendage of alkaline phosphatase signal to rat liver ΕR cytochrome b in plasmid constructions pAA-cyt, pSΕC-cyt/C and pSΕC-cyt/N. The full-length synthetic cytochrome b, gene, contained within the Psrl-EcoRl sites and carrying an in-built πbosomal binding site, was cloned within the polylinker site of pBluescript SK+ to give pBLUΕ-cyt. In step 1 , the unique Psrl site, located upstream of the Shine/Dalgarno sequence of cytochrome b 5 gene was displaced immediately adjacent to the cytochrome b methionine initiator codon by replacing Psrl-Aatll deletion with the 27bp synthetic oligonucleotide duplex. This gave plasmid pA-cyt. Icyt. In step 2, insertion of the modified β.//7/HI-H//?c/III-cleaved cytochrome b 5 gene (from pA-cyt) into identically-cleaved pFOG402. This yielded plasmid pAF-cyt in which cytochrome b 5 is placed under the control of phoA promoter but in an unphased reading frame with the alkaline phosphatase signal sequence. In step 3. involving construction of pAA-cyt, it shows the generation of the correct reading frame between the signal sequence and cytochrome b 5 by flush-ending Psrl-BamHl- cleaved PAF-cyt and recircularising the plasmid. In this process the 21 residue signal sequence was installed in a contiguous reading frame with the mammalian cytochrome b, such that the + 1 arginyl residue of mature alkaline phosphatase became included. This was to enhance the export properties of the cytochrome b 5 apo- protein. To minimise lethality to the host, the chimera was placed under tight transcriptional control of the native pho promoter in plasmid pAA-cyt, inducible by growth of the bacteria in a phosphate-limited medium. In step 4. the plasmid was further modified by appending the tail portion (T) of cytochrome b s . Finally . translocation of EcoO109-cleaved cytochrome bX segment into identically cleaved PUC 19 gave plasmid PSΕC-cyt/C. Plasmid PSEC-cyt/N was derived by excising EcoRl-Hindlll fragment from PAF-cyt containing pho- -ss-b- and cloning into identically cleaved pUC19. Referring now to Figure 8. an expression vector as illustrated in Figure 2 was modified by appending in fusion at the C-terminus of cytochrome b, with a gene sequence coding for the transit peptide ot nitrite reductase (step 1 ). The modified plasmid (pCYT- nTP) was introduced into the cytoplasmic region of E.coli TB-1 (Lac ) cells and the corresponding chimeπc protein comprising the cytochrome b, apo-protein fused at its C- terminus with nitrite reductase transit peptide (pre-form apo-form of cytochrome b,-nιtπte reductase transit peptide). was expressed in the cytoplasm (step 2). The pre-form chimeπc protein was processed (apo form ot cytochrome b,-nιtπte reductase transit peptide) and after being translocated into the penplasmic region ot the £ toll cells (step 3). it combined with the haem-prosthetic group to give the holo-protein (holo form of cytochrome b, -nitrite reductase transit peptide) The crude recombinant chimeπc protein was extracted from the pink bacteria (20ml culture) by an osmotic shock technique (step 4). The technique involved suspending the cells in 1ml of 20% sucrose. 0.3M Tris-HCl (pH 8), I mM Na 2 EDTA (STE) and incubation at room temperature tor ten minutes. The cells were harvested as pellet by centrifugation at 5,000g for six minutes and resuspended in residual STE volume. The plasmolysed cells were osmotically shocked by suspending in a minimum possible volume (approximately 5ml) of ice-cold 0.5mM MgCK and left on ice for 10 minutes, followed by centrifugation at lO.OOOg tor ten minutes to yield a supernatant comprising the penplasmic fraction of the cells. The chimera traced by its bright pink colour was purified by a single-step affinity chromatographv (step 5). such as DEAE Sepharose column chromatography or haem- affimty chromatography. The chimera was then selectively cleaved (step 6) by CNBr treatment to generate authentic nitrite reductase transit peptide. The 21 residue signal sequence is installed in a contiguous reading frame with the mammalian cytochrome b,, the + 1 arg yl residue of mature alkaline phosphatase being included to enhance the export properties of the apo-protein chimera. Referring now to Figure 9. an expression vector as illustrated in Figure 3 was modified by inserting in a contiguous reading between the signal sequence and cytochrome b,, a BamHl-Psrl synthetic gene coding tor nitrite reductase transit peptide (step 1). The derivative plasmid (pnTP-cyt) was introduced into the cytoplasmic region of E.coli TB- 1 (Lac ) cells and the corresponding chimeπc protein comprising the pre-form apo-form of nitrite reductase transit peptide-cvtochrome b, was expressed in the cytoplasm (step 2). The processed form of the chimeπc protein (apo-form of nitrite reductase transit peptide- cytochrome b s ) was then translocated into the penplasmic region of the E.coli cells (step 3), where combination with the haem-prosthetic group occurred constituting the holo-protein. The crude recombinant protein was extracted from the pink bacterial (20ml culture) by an osmotic shock technique (step 4). The technique involved suspending the cells in 1 ml of 20% sucrose. 0.3M Tπs-HCl (pH 8). I mM Na.EDTA (STE) and incubation at room temperature for ten minutes. The cells were harvested as pellet by centrifugation at 5.000g for 6 minutes and resuspended in residual STE volume. The plasmolysed cells were osmotically shocked by suspending in a minimum possible volume (approximately 5ml) of ice-cold 0.5mM MgCl 2 and left on ice for 10 minutes, followed by centrifugation at 10,000g for ten minutes to yield a supernatant comprising the periplasmic fraction of the cells. The chimera traced by its bright pink colour was purified by a single-step affinity chromatography (step 5), such as DEAE Sepharose column chromatography or haem- affinity chromatography. The nitrite reductase transit peptide was cleaved from the isolated chimera by CNBr. The 21 residue signal sequence is installed in a contiguous reading frame with the foreign gene and the subsequent mammalian cytochrome b,. The + 1 arginyl residue of mature alkaline phosphatase being included to enhance the export and processing characteristics of the chimeπc apo-protein.

Claims: Claims 1 A genetic precursor unit w Inch comprises a nucleotide sequence coding for a pre-form apo-protein, said nucleotide sequence being such that said pre-form apo-protein is translocatable from a cytoplasmic region ot a cell to the penplasmic region of said cell, so as to permit constitution in said penplasmic region of a processed apo-protein and conversion into a corresponding holo-protein - A genetic unit according to claim 1. which is suitable for expression in the cytoplasmic region ot an E tali cell 3. A genetic unit according to claim 2. wherein said E. coli cell comprises E.coli strain TB- 1 , [F ara Λ ( c-pioAB) rps o 80./ lacZλI.115 h .dRX (rk+ m + k)] or N4830- 1 [f- ,ιto ~- CI857 H I)] 4. A genetic unit according to any ot claims 1 to 3. wherein said apo-protein comprises a cytoplasmic cytochrome 5. A genetic unit according to claim 4. wherein said cytoplasmic cytochrome comprises the soluble core domain ot cytochrome b. of liver endoplasmic reticulum. 6. A genetic unit according to any ot claims 1 to 5, which comprises a nucleotide sequence which codes tor an ammo-terminal signal peptide capable of directing the pre-form apo-protein to the cv toplasmic membrane tor translocation into the peπplasm of the cell. 7. A genetic unit according to claim 5. which comprises a nucleotide sequence which codes for an amino-terminal signal peptide in linkage with cytochrome b-. 8. A genetic unit according to claim 6 or 7. wherein said signal peptide comprises E.coli alkaline phosphatase 9 A vector substantially as illustrated in any ot Figures 1 to 3 10. A DNA sequence substantially as illustrated in any of Figures 4 to 6 (SΕQ ID NOS 1 to 3 respectively) 11. An assay system for a signal peptidase. which assay system comprises: (a) a genetic precursor unit which comprises a nucleotide sequence coding for an apo-protein and an amino-terminal signal peptide. said nucleotide sequence being such that said apo-protein is translocatable from a cytoplasmic region of a cell to the periplasmic region of said cell, so as to permit constitution in said periplasmic region of a processed apo-protein: and (b) signal peptidase. a source thereof or a precursor therefor, wherein the signal peptidase can effect proteolytic cleavage of the amino terminal signal peptide from said apo-protein. 12. An assay system according to claim 1 1. wherein said genetic unit is suitable for expression in the cytoplasmic region of an E.coli cell. 13. An assay system according to claim 1 1 or 12. which further comprises a host medium for said genetic unit. 14. An assay system according to claim 13, wherein said host medium comprises E.coli. 15. An assay system according to claim 14, wherein said E.coli comprises E.coli strain TB- 1 , [F ara Δ (\ac-proAB) rps φ 80^ lacZAM15 hsclR17 (rk+ m + k)} or N4830-1 [f- SIIO his-i/v-galK-(\chl D-pgJ )(\Ba Λ'+ C1857 H I )]. 16. An assay system according to any of claims 1 1 to 15. wherein said apo-protein comprises a cytoplasmic cytochrome. 17. An assay system according to claim 16, wherein said cytoplasmic cytochrome comprises the soluble core domain of cytochrome b of liver endoplasmic reticulum. 18. An assay system according to any of claims 1 1 to 17, which comprises a nucleotide sequence which codes for an amino-terminal signal peptide in linkage with cytochrome b s . 19. An assay system according to any of claims 1 1 to 18, wherein said signal peptide comprises E.coli alkaline phosphatase. 20. An assay system according to any of claims 1 1 to 19, which further comprises means for monitoring the proteolvtic cleavage of the am o terminal signal peptide from the apo-protein. ~ 1. An assay system according to claim 20. wherein said monitoring means comprises means for separating the cleaved apo-protein. and means for spectrophotometrically monitoring the cleaved apo-protein following supplementation thereof with exogenous haem. 22. An assay system according to claim 21 , wherein the pre-form apo-protein is phase- separated from the cleaved apo-protein. 23. A method of identifying inhibitors to signal peptidase, which method comprises providing an assay system according to any of claims 1 1 to 22, introducing a test material into cells of a host medium used in said assay system, and monitoring the effect of said test material on a selected activity of said signal peptidase. 24. A method according to claim 23, which involves spectrophotometrically monitoring the cleavage of amino-terminal signal peptide from the apo-protein. 25. A method according to claim 23 or 24. wherein the test material comprises any of consensus signal peptide. one or more transition metal ions and a carboxy-modifying reagent. 26. A test material identified as an inhibitor to signal peptidase by a method according to any of claims 23 to 25, for use in modulating in vivo protein secretion. 27. A test material according to claim 26. which comprises a synthetic inhibitor of signal peptidase. 28. Λ method of protein synthesis hich comprises provision of a genetic unit comprising a nucleotide sequence coding tor a pre-form of an apo-protein, synthesizing said pre¬ form apo-protein in a cytoplasmic region ot a cell, and translocating said synthesised pre-form apo-protein to the penplasmic region of said cell, so as to permit constitution in said penplasmic region of a signal-processed apo-protein which can then be converted to a corresponding holo-protein. 29. A method according to claim 28. w nerein said genetic unit is as defined in any of claims 1 to 8. 30. A method according to claim 28 or 29. wherein said cell comprises an E.coli cell. 31. A method according to claim 30. w herein said E.coli compπses E.coli strain TB-1 , [F ara Δ (lac-proAB) rps φ 80./ lacZ M15 hsclRX (rk+ m ~ k)] or N4830-1 [f-suo his- 32. A method according to any of claims 28 to 31 , which compπses initiation of transcription of said genetic unit by control ot the incubation conditions of a host medium. 33. A method according to claim 32, wherein said initiation comprises cultivation of a bacterial host in a phosphate-limited medium 34. A method according to any ot claims 28 to 33. which comprises spectrophotometnc or visual monitoring of a characteristic colour change of host bacterial cells which is indicative of the synthesis of said holo-protein or its fusion counterpart in said bacterial cells. 35. A method according to any ot claims 28 to 34. which further compπses the step of directing the apo-protein to the cytoplasmic membrane, under the direction of a signal peptide. so as to facilitate translocation of the pre-form apo-protein from the cytoplasm to the penplasmic space ot the cell 36. A method according to any of claims 28 to 35. which compπses translocation of a prosthetic group from the cytoplasmic region of the cell to the periplasmic region, for combination with the signal-processed apo-protein in the periplasmic region to form the holo-protein. 37. A method according to claim 36. wherein the apo-protein is translocated to the periplasmic region, followed by translocation of the prosthetic group, such that the apo-protein can act as a sink for the latter. 38. A method according to claim 36 or 37. wherein the apo-protein comprises the cytochrome b, soluble core, and the prosthetic material comprises the haem prosthetic group. 39. A method according to any of claims 28 to 38, wherein synthesis of the holo-protein is at a level of at least 6 mg/litre of culture after five hours induction. 40. A proteinaceous material comprising a pre-form apo-protein which is synthesized in the cytoplasmic region of a cell, the pre-form apo-protein being translocatable to the periplasmic region of said cell, so as to allow constitution in said periplasmic region of a correspondingly processed holo-protein. 41. A proteinaceous material according to claim 40. wherein said apo-protein is in combination with a signal peptide for selective translocation or secretion of said apo- protein from the cytoplasmic to the periplasmic region of a cell, so as to allow constitution in said periplasmic region of a corresponding holo-protein from said apo- protein. 42. A proteinaceous material according to claim 41. wherein said signal peptide comprises E.coli alkaline phosphatase. 43. A proteinaceous material according to any of claims 40 to 42. wherein said apo- protein comprises a cytoplasmic cytochrome. 44. A proteinaceous material according to claim 43. wherein said cytoplasmic cytochrome comprises the soluble core domain of cytochrome b of liver endoplasmic reticulum. 45. A holo-protein constituted in the periplasmic region of a cell from an apo-protein of a proteinaceous material according to any of claims 41 to 44. 46. A holo-protein derived from a genetic precursor unit substantially as illustrated with reference to any of Figures 4 to 6 (SEQ ID NOS 1 to 3, respectively). 47. A kit for use in protein synthesis, which kit comprises: (a) a bacterial cellular medium; and (b) genetic material comprising a nucleotide sequence coding for a pre-form apo-protein, such that said pre-form apo-protein coded for by said nucleotide sequence is translocatable from the cytoplasmic to the periplasmic region of a cell of said host medium, so as to allow constitution in said periplasmic region of a holo-protein from said apo-protein. 48. A kit according to claim 47, wherein said bacterial medium comprises E.coli.

Inventors: KADERBHAI MUSTAK ALI, KADERBHAI, MUSTAK, ALI
Applicants: KADERBHAI MUSTAK ALI, UNIV WALES, KADERBHAI, MUSTAK, ALI, UNIVERSITY COLLEGE OF WALES, ABERYSTWYTH
Application year: 1994
Main classification: C12N15
IPC codes: C07K14, C12N9, C12Q1